Live attenuated infectious laryngotracheitis virus (iltv) vaccines and preparation thereof

ABSTRACT

This disclosure describes a modified live infectious laryngotracheitis virus (ILTV) developed from a strain of ILTV grown at a reduced incubation temperature, vaccines that include the modified live ILTV, methods for producing the live modified ILTV, and methods that include administering the modified ILTV to a subject.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/896,729, filed Oct. 29, 2013, which is incorporated hereinby reference.

GOVERNMENT FUNDING

This invention was made with government support under 2013-31100-0610awarded by the U. S. Department of Agriculture. The government hascertain rights in the invention.

SUMMARY

This disclosure describes, in one aspect a modified live infectiouslaryngotracheitis virus (ILTV) developed from a strain of ILTV grown ata reduced incubation temperature. The ILTV strain may be of tissueculture origin (TCO) or chick embryo origin (CEO). In particularembodiments, the strain of ILTV can be tissue culture origin (TCO). Themodified live strain is serially passaged at a temperature in the rangeof 28° C. to 31° C., which is lower than the typical growth temperature(37° C.) for producing ILTV, for about 30-50 passages through a suitableculture medium. In some embodiments, the modified strain is generated byserially passaging at a temperature of 30° C. In some embodiments, thestrain is produced through 34-44 serial passages. In some embodiments,the culture medium may be chicken embryos or cell culture. In some ofthese embodiments, the culture medium is cell culture.

In some embodiments, the attenuated ILTV can possess at least onegenetic modification compared to the starting ILTV and/or a wild-typeILTV. In some embodiments, the genetic modification can include deletionof a thymine in the coding region for UL50. Such a deletion can resultin a frame shift at Asn10 of UL50.

In another aspect, this disclosure describes a vaccine for poultry. Thevaccine includes an effective amount of a modified live ILTV. Thevaccine may further include a pharmaceutically acceptable diluent,carrier, and/or adjuvant. The vaccine may be useful for immunizingpoultry. In some embodiments, the poultry can include a chicken. Thevaccine may be formulated for administering to poultry by any suitableroute. In some embodiments, the vaccine may be administered via an oral,an ocular (eye drop), an intranasal, an aerosol (spray), or an ingestion(drinking water) route. In particular embodiments, the vaccine may beadministered via an aerosol (spray) or drinking water administration,which can be more cost effective for commercial poultry production thanother routes of administration. In some embodiments, oral administrationof the modified live ILTV to a subject produces increased weight gainand/or decreased sign of infectious laryngotracheitis disease followingchallenge with a virulent ILTV, compared with an unvaccinated controlsubject.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. ILTV lesions, also referred to as plaques or pocks, on thechorioallantoic membrane (CAM) of the embryonating chicken egg at fivedays following inoculation. (A) Appearance of unique ILTV lesions on theCAM following inoculation with clone 5-7. Note the small well definedplaques. (B) Appearance of a typical ILTV plaque on the CAM. Notediffuse spreading lesion following inoculation with experimental vaccineP32 37C.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The disclosure relates to live attenuated infectious laryngotracheitisviruses (ILTVs) and the use of the live attenuated ILTVs as vaccines forpoultry. Poultry such as, for example, broiler chickens, vaccinated withthe vaccine may have a lower incidence of clinical disease signs and/orincreased growth (as measured, for example, by greater weight gain),than poultry vaccinated with chick embryo origin (CEO) vaccines. Also,poultry vaccinated using the methods and vaccines described herein canhave improved weight gain compared an unvaccinated poultry after beingchallenged with a virulent challenge strain of ILTV.

Modified live vaccines of two types have been available for many yearsto control infectious laryngotracheitis (ILT): vaccines of chick embryoorigin (CEO) and vaccines of tissue culture origin (TCO). The CEOproducts often exhibit negative side effects such as, for example,causing disease, reducing growth rates (exhibited as reduce weightgain), and reducing feed efficiency. Because of the side effect producedby the CEO vaccines, poultry producers may prefer not to use them unlessit becomes absolutely necessary to do so. Thus, many flocks gounvaccinated and are susceptible to the disease.

There are limited options for commercially available TCO vaccines. A TCOproduct is typically milder than a CEO vaccine, but is typicallyadministered via the ocular route. This severely limits its practicalcommercial use by poultry producers because the vaccine must beinstilled into the conjunctiva of each individual bird of a flock. This“eye drop” application can be laborious and expensive. Thus, poultryproducers disfavor the process and, consequently, TCO vaccines.

More recently, a third type of vaccine, a recombinant ILTV vaccine, hasbeen commercialized. A recombinant ILTV product can express one or moreimmunogenic proteins, but not the complete virus, and thus fail toproduce side effects associated with CEO vaccines. A disadvantage ofrecombinant vaccines, however, is that they can be expensive to purchasecompared to live vaccines, and are only partially effective. If a saferand effective live vaccine could be developed, the cost of vaccineproduction would dramatically reduce costs to control ILT.

The new vaccine described herein can replace the use of existingmodified live and recombinant vaccines. For example, in commercial usewith broiler chicken production, a vaccine prepared with the liveattenuated ILTV described herein can offer a cheaper alternative torecombinant vaccines and be safer than current modified live vaccines.As another example, in commercial production of breeder chickens and/orlayer chickens, the live attenuated ILTV described herein can be acomponent of a primary vaccine could be used for primary vaccination,which can be followed with one or more booster vaccinations using, forexample, currently available CEO vaccines.

In one embodiment, a commercially available vaccine referred to as atissue culture origin (TCO) vaccine (LT-IVAX, Merck Animal Health,Summit, N.J.), was serially passaged up to approximately 40 times insuccessive cultures of chicken kidney cells (CKC) in cell culturemaintained at 30° C., an incubation temperature lower than the typicalculture temperature used to grow ILTV (37° C.). The virus was thencloned in CKC culture at 30° C. by limiting dilution to obtain a morehomogeneous virus population than is typically produced without limitingdilution. The more homogeneous virus population can produce moreconsistent vaccine characteristics when administered to poultry.

Most poultry vaccines are grown at 37° C. As just discussed above, theILTV described herein (P32 30C) was produced serially passaging thevirus in culture at a temperature of 30° C. A corresponding ILTV (P3237C) was prepared similarly to the P32 30C ILTV except that it wasgenerated by serially passaging the virus in a culture maintained at 37°C. The resulting experimental vaccines were tested in chickens and foundto be safe and efficacious by the ocular/eye drop and oral routes ofadministration.

TABLE 1 Post-vaccination assessment of ILTV commercial and experimentalvaccines in two-week-old broiler chickens vaccinated by the ocular ororal routes. Weight Gain From 1-6 Days Post Vaccination Clinical DiseaseAs a % of Non-Vacc. Signs Associated Group Treatment Weight Gain WithVaccination Combined Bodyweight & Clinical Disease Signs Data FollowingOcular or Oral Vaccination 1 TCO^(A) Commercial 94 6/15 2 CEO^(B)Commercial 92 12/15  3 P32 37 C.^(C) 87 4/15 4 P32 30 C.^(D) 92 1/15 5No Vaccine 100 0/15 Bodyweight & Clinical Disease Signs Data Post OcularVaccination 1 TCO Commercial 89 2/5  2 CEO Commercial 93 5/5  3 P32 37C. 105 1/5  4 P32 30 C. 103 0/5  5 No Vaccine 100 0/5  Bodyweight &Clinical Disease Signs Data Post Oral Vaccination 1 TCO Commercial 964/10 2 CEO Commercial 92 7/10 3 P32 37 C. 78 3/10 4 P32 30 C. 87 1/10 5No Vaccine 100 0/10 ^(A)TCO = Tissue culture origin (LT-IVAX, MerckAnimal Health, Summit, NJ). ^(B)CEO = Chicken embryo origin (TRACHIVAX,Merck Animal Health, Summit, NJ). ^(C)P32 37 C. = 32 tissue culturepassages performed at 37° C. ^(D)P32 30 C. = 32 tissue culture passagesperformed at 30° C.

Vaccination with commercial (TCO Commercial and CEO Commercial) andexperimental (P32 37C and P32 30C) vaccines exhibited somewhat reducedgrowth of broiler chickens as measured by bodyweight gain (Table 1). Thenegative effect of vaccination on bodyweight gain is a general drawbackto the use of modified live vaccines for controlling ILTV infections.The negative effect on bodyweight gain is typically offset by improvedhealth of vaccinated birds compared to non-vaccinated birds beyond theperiod reflected in Table 1.

Another disadvantage to using modified live vaccines is their tendencyto produce clinical disease signs following vaccination. The P32 30Cvaccine produced the fewest number of chickens with clinical diseasesigns. Clinical disease signs include, for example, conjunctivalswelling and/or reddening, presence of an ocular and/or nasal (nares)exudate, nasal sinus swelling, labored breathing, and/or coughing, andin severe cases, a bloody expectoration.

TABLE 2 Post-vaccination assessment of ILTV commercial and experimentalvaccines in two-week-old broiler chickens vaccinated by the ocular ororal routes. Q-PCR and ILT Serum Antibody Data Following Ocular or OralVaccination ILTV Serology-Day 14 Q-PCR Values-Day 6 Post VaccinationPost Vaccination ILTV #of Chickens Total Group Antibody with PositiveGroup Treatment Combined Ocular Oral Titers Titer/Total 1 TCO Commercial31.6 35.2 29.8 184 3/15 2 CEO Commercial 29.0 27.3 29.9 1003 12/15  3P32 37C 33.2 34.1 35.9 1301 12/15  4 P32 30C 36.5 39.4 35.1 15 6/15 5 NoVaccine 46.0 46.0 46.0 0 0/15

Quantitative (Q)-PCR values are an indication of the presence of ILTVvaccine in the oral/pharyngeal cavity, a body site where the vaccine maybe detected. Values are inversely related to virus levels; Lower Q-PCRvalues equate to the presence of higher virus genome levels. Regardlessof the type of vaccine tested, ILTV was not detected in great quantitieson Day 6 post-vaccination.

The findings indicate that chickens given the CEO Commercial vaccine hadthe highest levels of ILTV-specific genome based on Q-PCR. A differenceof 3.0 between Q-PCR values represents a 10-fold difference in the levelof detectable viral genome. Chickens vaccinated with the P32 30C vaccineshowed the lowest level of ILTV in the oral/pharyngeal cavity,suggesting that the vaccine is more attenuated than the other vaccinestested.

Serum antibody titers for ILTV were measured on day 14 post-vaccinationusing a commercial ELISA kit (Synbiotics Corp., Kansas City, Mo.). TheCEO Commercial and P32 37C vaccines induced the highest antibody titers,detected in 12/15 chickens. This finding, along with the higherincidence of clinical disease signs and weight suppression suggest thatthe CEO Commercial and P32 37C experimental vaccines were notattenuated—i.e., they were more invasive than the TCO Commercial and P3230C vaccines. Moreover, ELISA serum antibody titers may not correlatewell with protection (Fahey et al. 1990. J. Gen. Virol. 71:2401-2405).Thus, a more attenuated vaccine like the P32 30C vaccine can induceprotection (Table 3) even when producing low serum antibody titers.

TABLE 3 Post-challenge assessment of ILTV commercial and experimentalvaccines in two-week-old broiler chickens vaccinated by the ocular ororal routes. Weight Gain From 1-6 Birds with Days Post ChallengeClinical Disease Microscopic As a % of Non-Vacc. Signs AssociatedPathology Group Treatment Weight Gain With ILTV Infection Eyelid TracheaBodyweight and Protection (Clinical Signs and Microscopic Pathology)Data After Challenge of Chickens Vaccinated via the Ocular or OralRoutes-Combined Data 1 TCO Commercial 678 0/13  0/13  0/13 2 CEOCommercial 711 0/13  0/13  0/13 3 P32 37 C. 677 0/13  0/13  0/13 4 P3230 C. 741 0/12  0/13  0/13 5 No Vaccine 100 12/12  12/12  5/12Bodyweight and Protection (Clinical Signs and Microscopic Pathology)Data After Challenge of Chickens Vaccinated via the Ocular Route 1 TCOCommercial 618 0/5 0/5 0/5 2 CEO Commercial 704 0/5 0/5 0/5 3 P32 37 C.626 0/5 0/5 0/5 4 P32 30 C. 716 0/5 0/5 0/5 5 No Vaccine 100 12/12 12/12  5/12 Bodyweight and Protection (Clinical Signs and MicroscopicPathology) Data After Challenge of Chickens Vaccinated via the OralRoute 1 TCO Commercial 716 0/8 0/8 0/8 2 CEO Commercial 715 0/8 0/8 0/83 P32 37 C. 708 0/8 0/8 0/8 4 P32 30 C. 753 0/7 0/8 0/8 5 No Vaccine 10012/12  12/12  5/12

All vaccines induced protection as measured by weight gain from days 1-6after challenge, the lack of clinical disease signs, and microscopicpathology observed after ocular and intratracheal route challenge withthe virulent USDA challenge strain of ILTV. The Animal and Plant HealthInspection Service of the USDA provides the challenge strain toresearchers for the purpose of evaluating immunity of chickens followingvaccination with experimental and/or commercial vaccines. It is thus a“standard” strain used for challenge purposes.

Chickens vaccinated with the P32 30C vaccine showed the greatest weightgain, indicating that the broilers were protected. Clinical diseasesigns observed only in the broilers in the No Vaccine treatment includedconjunctival swelling and reddening, presence of an ocular and/or nasal(nares) exudate, labored breathing and coughing. Microscopic pathologyof eyelid and trachea was performed to determine whether clinical signsof ILTV infection associated with the virulent challenge were present inthese tissues.

Thus, the data in Tables 1-3 demonstrate that the P32 30C vaccine ismore attenuated than the other vaccines, based on its ability to producemilder clinical signs. The P32 30C vaccine, however, produced bodyweight suppression when administered by the oral route, the most commonmethod of vaccine application in commercial practice. For this reason,we decided to clone the P32 30C vaccine using a limit dilutionprocedure. The limit dilution procedure (Example 2) produced Clone 5-7.Clone 5-7 was evaluated and the results are provided in Tables 4-6.Three serial ten-fold dilutions (10⁻¹, 10⁻², and 10⁻³) of the virusstock were used to vaccinate broiler chickens at two weeks of age.Vaccines were administered by the ocular or oral route.

TABLE 4 Post-vaccination assessment of varying dilutions (10⁻¹-10⁻³) ofILTV experimental vaccine Clone 5-7 in two-week-old broiler chickensvaccinated by the ocular or oral route. Weight Gain From 1-6 Days PostVaccination Clinical Disease as a % of Non-Vacc. Signs Associated GroupTreatment Weight Gain With Vaccination Combined Bodyweight DataFollowing Ocular or Oral Vaccination 1 Clone 5-7 94  2/10 Dilution 10⁻¹2 Clone 5-7 92  0/12 Dilution 10⁻² 3 Clone 5-7 101  0/12 Dilution 10⁻³ 4No Vaccine/ Not Applicable Not Applicable Challenge 5 No Vaccine/ 100Not Applicable No Challenge Bodyweight Data Post Ocular Vaccination 1Clone 5-7 103 0/5 Dilution 10⁻¹ 2 Clone 5-7 94 0/5 Dilution 10⁻² 3 Clone5-7 100 0/5 Dilution 10⁻³ 4 No Vaccine/ Not Applicable Not ApplicableChallenge 5 No Vaccine/ 100 Not Applicable No Challenge Bodyweight DataPost Oral Vaccination 1 Clone 5-7 85 2/5 Dilution 10⁻¹ 2 Clone 5-7 860/5 Dilution 10⁻² 3 Clone 5-7 101 0/5 Dilution 10⁻³ 4 No Vaccine/ NotApplicable Not Applicable Challenge 5 No Vaccine/ 100 Not Applicable NoChallenge

Clone 5-7 dilutions given ocularly had minimal impact on chickens 1-6days post vaccination. Oral administration reduced weight gain at the10-1 and 10-2 dilutions, but not at the 10-3 dilution. Clinical diseasesigns were only observed at the highest oral dose (10⁻¹ dilution.

Chickens receiving the 10⁻³ dilution of the vaccine were free ofclinical signs and gained bodyweight at the same rate as chickens notreceiving the vaccine or the virulent challenge (“No Vaccine/NoChallenge” treatment). Thus, Clone 5-7 demonstrated the potential to bea safe and economically desirable vaccine candidate.

TABLE 5 Post-vaccination assessment of dilutions 10⁻¹ to 10⁻³ of ILTVexperimental vaccine Clone 5-7 in two-week-old broiler chickensvaccinated by the ocular or oral routes. LT Antibody Serology- Day 14Post Vaccination # of Birds Q-PCR Values- Day 6 ELISA w/Positive # ofBirds # of Birds Post Vaccination Titers: Titer: ELISA w/Positive ELISAw/Positive Combined Combined Combined Titers: Titer: Titers: Titer:Group Treatment Group Ocular Oral Group Group Ocular Ocular Oral Oral 1Clone 5-7 Dilution 10⁻¹ 39.2 41.8 36.7 894 6/9  451 2/4 1211 4/5 2 Clone5-7 Dilution 10⁻² 37.4 40.9 32.7 786 9/12 584 4/7 1189 4/5 3 Clone 5-7Dilution 10⁻³ 37.8 37.0 41.4 368 6/12 334 2/7 295 2/5 4 NoVaccine/Challenge Not Not Not 17 0/14 17  0/14 17  0/14 DetectedDetected Detected 5 No Vaccine/No Challenge N/A N/A N/A N/A N/A  N/A N/AN/A N/A N/A = Not Applicable

Again, quantitative (Q)-PCR values are an indication of the presence ofILTV vaccine in the oral/pharyngeal cavity, a site where the vaccine maybe detected. Values are inversely related to virus genome levels; LowerQ-PCR values equate to the presence of higher virus genome levels.Regardless of the type of vaccine tested, ILTV was not detected in greatquantities on Day 6 post-vaccination. A difference of 3.0 between Q-PCRvalues represents a 10-fold difference in the level of detectable viralgenome. Chickens vaccinated with the dilutions of the Clone 5-7 vaccineshowed a low level of ILTV in the oral/pharyngeal cavity suggesting thevaccine is attenuated.

Serum antibody titers for ILTV were measured on day 14 post-vaccinationusing a commercial ELISA kit (Synbiotics Corp., Kansas City, Mo.). Fiftypercent or more of chickens vaccinated with the Clone 5-7 vaccine viathe ocular or oral route (see Combined Group) produced an ILTV serumantibody response. In each vaccine dilution treatment group, ILTVantibodies were detected in the majority of birds. The data show agreater antibody response in birds receiving the 10⁻¹ and 10⁻² dilutionvaccines orally compared to chickens given the vaccines via the ocularroute. Chickens not given the vaccine (No Vaccine/Challenge treatment)did not produce ILTV serum antibody responses. Once again, however,ELISA titers may not correlate well with protection (Fahey et al. 1990.J. Gen. Virol. 71:2401-2405).

TABLE 6 Post-challenge assessment of dilutions 10⁻¹ to 10⁻³ of ILTVClone 5-7 vaccine in two-week-old broiler chickens vaccinated by theocular or oral route. Weight Gain From 1-6 Days Post VaccinationClinical Disease As a % of Non-Vacc. Signs Associated Group TreatmentWeight Gain With ILTV Infection Combined Bodyweight & Clinical DiseaseSigns Data Following Ocular or Oral Vaccination 1 Clone 5-7 99 1/8Dilution 10⁻¹ 2 Clone 5-7 92  0/10 Dilution 10⁻² 3 Clone 5-7 105 1/8Dilution 10⁻³ 4 No Vaccine/ 61 5/5 Challenge 5 No Vaccine/ 100  0/10 NoChallenge Combined Bodyweight & Clinical Disease Signs Data FollowingOcular Vaccination 1 Clone 5-7 123 1/3 Dilution 10⁻¹ 2 Clone 5-7 94 0/5Dilution 10⁻² 3 Clone 5-7 140 1/4 Dilution 10⁻³ 4 No Vaccine/ 61 5/5Challenge 5 No Vaccine/ 100  0/10 No Challenge Combined Bodyweight &Clinical Disease Signs Data Following Oral Vaccination 1 Clone 5-7 970/5 Dilution 10⁻¹ 2 Clone 5-7 106 0/5 Dilution 10⁻² 3 Clone 5-7 99 0/4Dilution 10⁻³ 4 No Vaccine/ 61 5/5 Challenge 5 No Vaccine/ 100  0/10 NoChallenge

After challenge with virulent USDA challenge strain of ILTV, chickensreceiving any ILTV Clone 5-7 vaccine dilution had higher weight gainthan non-vaccinated/challenged chickens (No Vaccine/Challengetreatment). Following challenge, chickens vaccinated via the ocular ororal routes had fewer clinical disease signs compared to chickens notreceiving a vaccination. All chickens vaccinated via the ocular routewere protected against clinical disease signs after challenge.

TABLE 7 Post-vaccination assessment of 10⁻² dilution of ILTVexperimental vaccine Clone 5-7 in five-week-old specific pathogen-free(SPF) chickens vaccinated by the ocular route. Passage 1 and Passage 2.Clinical Disease Signs Observed Bird During 14 Days Q-PCR Ct Value:Number Vaccinated Before Challenge Oral Swabs 5 DPV Passage 1. OralVaccine Shed Following Ocular Vaccination Y41 Yes None 32.7* Y42 YesNone 32.5* Y43 Yes None 33*  Y44 Yes None Undetermined Y45 Yes None35.3  Y46 Yes None 34.5  Y47 Yes None 30.1* Y48 Yes None 35.0  Y49 YesNone 32.4* Y50 Yes None Undetermined Passage 2. Oral Vaccine ShedFollowing Ocular Vaccination G61 Yes None Undetermined G62 Yes NoneUndetermined G63 Yes None Undetermined G64 Yes None Undetermined G65 YesNone Undetermined G66 Yes None Undetermined G67 Yes None UndeterminedG68 Yes None Undetermined G69 Yes None Undetermined G70 Yes NoneUndetermined Inoculum Ct Value of 28 (Clone 5-7 diluted 10⁻² or 1:100).*Used in pool for Passage 2 inoculum.

In Passage 1, five days post ocular vaccination with the ILTV Clone 5-7vaccine, minimal shedding was observed in oral swabbings. In Passage 2,virus was not detected five days post vaccination with materialcollected from Passage 1 birds.

TABLE 8 Post-challenge assessment of 10⁻² dilution of ILTV experimentalvaccine Clone 5-7 in five-week- old specific pathogen-free (SPF)chickens vaccinated by the ocular route. Passage 1 and Passage 2. BirdNum- Clinical Disease Signs Observed: Days Post Challenge ber Vacc. 1 23 4 5 6 7 8 9 10 Passage 1. Post Challenge Data Y41 Yes None None NoneNone None None None None None None Y42 Yes None None None None None NoneNone None None None Y43 Yes None None None None None None None None NoneNone Y44 Yes None None None None None None None None None None Y45 YesNone None None None None None None None None None Y46 Yes None None NoneNone None None None None None None Y47 Yes None None None None None NoneNone None None None Y48 Yes None None None None None None None None NoneNone Y49 Yes None None None None None None None None None None Y50 YesNone None None None None None None None None None B26 No None None NoneWet Eye (+1)^(A )  Conj. (+3)^(B) Conj. (+3)  Wet Eye (+1)   None NoneNone B27 No None None None Wet Eye (+1)   Conj. (+2)  Wet Eye (+1)  None None None None B28 No None None None Wet Eye (+1)   None Wet Eye(+1)   None None None None B29 No None None None Wet Eye (+1)   Conj.(+3)  Conj. (+3)  None None None None B30 No None None None Wet Eye(+1)   None None None None None Normal Passage 2. Post Challenge DataG61 Yes None None None None  Conj. (NS)^(C) Conj. (NS) Conj. (NS) NoneNone None G62 Yes None None None Wet eye (+1),  Conj. (NS) Conj. (NS)None None None None NE^(A) G63 Yes None None None None Conj. (NS) Conj.(NS) None None None None G64 Yes None None None  Conj. (+1)^(B) Conj.(NS) Conj. (NS) Conj. (NS) None None None G65 Yes None None None Wet eye(+1)   Conj. (NS) Conj. (NS) None None None None G66 Yes None None NoneConj. (+1) Conj. (NS) Conj. (NS) None None None None G67 Yes None NoneNone None Conj. (NS) Conj. (NS) None None None None G68 Yes None NoneNone Conj. (+1) Conj. (NS) Conj. (NS) None None None None G69 Yes NoneNone None Wet eye Conj. (NS) Conj. (NS) None None None None G70 Yes NoneNone None Conj. (+2) Conj. (NS) Conj. (NS) Conj. (NS) None None None P16No None None None Conj. (+1) Conj. (NS) Conj. (NS) Conj. (NS) Conj. (NS)Wet eye (NS) None P17 No None None None Wet eye (+1)   Conj. (NS) Conj.(NS) None None None None P18 No None None None None Conj. (NS) Conj.(NS) None None None None P19 No None None None None Conj. (NS) Conj.(NS) None None None None P20 No None None None Conj. (+2) Conj. (NS)Conj. (NS) Conj. (NS) None None None ^(A)Observed clinical disease signs(severity of clinical disease sign: +1 = mild; +2 = moderate; +3 =severe). ^(B)Conj. = Conjunctivitis ^(C)NS = Not Scored

In Passage 1, following challenge with the ILTV USDA challenge strain,all chickens vaccinated via the ocular route with ILTV Clone 5-7 wereprotected against clinical disease signs compared to chickens notreceiving a vaccination. In Passage 2, there was no difference betweenbirds vaccinated with the oral swab material collected in Passage 1 andthe non-vaccinated birds. All birds in Passage 2 showed clinical diseasesigns compatible with ILTV induced disease. These results show that theILTV Clone 5-7 is able to induce protective immunity (Passage 1) anddoes not spread to naïve susceptible chickens.

Thus, this disclosure describes an attenuated infectiouslaryngotracheitis virus (ILTV) that may be used as a component in avaccine to immunize a subject against infection by a virulent infectiouslaryngotracheitis virus. Generally, the attenuated ILTV can be producedby serial passage at a temperature that is reduced from the temperatureat which the ILTV is typically grown. Once the attenuated ILTV is sogenerated, however, one can generate the attenuated ILTV usingconventional molecular biology techniques to produce a virus having thesame nucleotide and/or functional features of the attenuated ILTVproduced as described herein.

While described herein in the context of an exemplary embodiment inwhich the attenuated ILTV is produced by serial passaging an ILTV virus,the attenuated ILTV described herein—and variants thereof—can beproduced by any suitable method. Also, while expressly exemplifiedherein in the context of an exemplary embodiment in which the attenuatedILTV possesses each of the genetic modifications listed in Table 11, anattenuated ILTV as described herein may contain fewer than all of thegenetic mutations listed in Table 11 and still retain an attenuatedcharacter.

In some embodiments, the attenuated ILTV can be produced by serialpassage of the virus at a minimum temperature of at least 25° C. suchas, for example, at least 27° C., at least 28° C., at least 29° C., atleast 30° C., at least 31° C., or at least 32° C. In some embodiments,the attenuated ILTV can be produced by serial passage of the virus at amaximum temperature of no more than 35° C. such as, for example, no morethan 34° C., no more than 33° C., no more than 32° C., no more than 31°C., no more than 30° C., or no more than 29° C. In some embodiments, theattenuated ILTV can be produced by serial passage of the virus at atemperature within a range having endpoints defined by any minimumtemperature listed above and any maximum temperature that is greaterthan the minimum temperature. For example, in one embodiment, theattenuated virus can be produced by serial passage of virus at atemperature of from 28° C. to 31° C. In one particular embodiment, theattenuated ILTV can be produced by serial passage of the virus at atemperature of 30° C.

The attenuated ILTV can be generated by any suitable number of serialpassages. In some embodiments, the attenuated ILTV can be produced by amethod that includes a minimum of at least two serial passages of thevirus such as, for example, at least three serial passages, at leastfive serial passages, at least seven serial passages, at least 15 serialpassages, at least 25 serial passages, at least 30 serial passages, atleast 34 serial passages, at least 40 serial passages, or at least 45serial passages. In some embodiments, the attenuated ILTV can beproduced by a method that includes a maximum of no more than 50 serialpassages such as, for example, no more than 44 serial passages, no morethan 30 serial passages, no more than 20 serial passages, no more than10 serial passages, no more than seven serial passages, no more thanfive serial passages, no more than four serial passages, or no more thanthree serial passages. In some embodiments, the attenuated ILTV can beproduced by a method that includes a number of serial passages within arange having endpoints defined by any minimum number of passages listedabove and any maximum number of serial passages that is greater than theminimum number of serial passages. In one particular embodiment, theattenuated ILTV may be generated using a method that includes sevenserial passages of the virus. Thus, in some embodiments, the attenuatedvirus can be produced by a method that includes, for example, 30-50serial passages such as, for example, 34-44 serial passages.

The serial passaging of the virus at a reduced temperature produces anattenuated virus that includes one or more genetic modificationscompared to the ILTV that was the starting material. In the exemplaryembodiment described above, the starting ILTV was a commerciallyavailable ILTV of tissue culture origin, LT-IVAX (Merck Animal Health,Summit, N.J.). Table 11 lists genetic modifications of the Clone 5-7generated as described herein compared to the LT-IVAX. Some of themutations are located in non-coding regions. Others, however, introduceframe shift mutations or amino acid substitutions

As noted above, while exemplified herein in the context of an exemplaryembodiment in which the attenuated ILTV possesses each of the geneticmodifications listed in Table 11, the attenuated ILTV contemplatedherein may contain fewer than all of the genetic mutations listed inTable 11 and still retain an attenuated character. Exemplary geneticmodifications can produce, for example, a frame shift mutation in one ormore coding regions of the viral genome. Exemplary coding regions thatmay be affected by a frame shift mutation include, for example, ORF B,ORF F, UL50, UL7, or UL9.

A frame shift mutation in ORF B may include, for example, a frame shiftintroduced at Phe54 such as may be produced by a deletion of anucleotide or an addition of a nucleotide in the codon for Phe54 of ORFB. One exemplary mutation is a deletion of a thymine (e.g., the thymineat nucleotide position 23030 of the ILTV genome) from the codon, but aperson of ordinary skill in the art can readily conceive of othermutations that could cause a frame shift at Phe54—or any other site—ofORF B.

A frame shift mutation in ORF F may include, for example, a frame shiftintroduced at Glu233 such as may be produced by a deletion of anucleotide or an addition of a nucleotide in the codon for Glu233 of ORFF. One exemplary mutation is an addition (e.g., a guanine added betweennucleotide position 5377 and 5378 of the ILTV genome) in the codon, buta person of ordinary skill in the art can readily conceive of othermutations that could cause a frame shift at Glu233—or any other site—ofORF F.

A frame shift mutation in UL50 may include, for example, a frame shiftintroduced at Asn10 such as may be produced by a deletion of anucleotide or an addition of a nucleotide in the codon for Asn10 ofUL50. One exemplary mutation is a deletion of a thymine (e.g., adeletion of the thymine at nucleotide position 15577 of the ILTV genome)from the codon, but a person of ordinary skill in the art can readilyconceive of other mutations that could cause a frame shift at Asn10—orany other site—of UL50.

A frame shift mutation in UL7 may include, for example, a frame shiftintroduced at Phe104 such as may be produced by a deletion of anucleotide or an addition of a nucleotide in the codon for Phe104 ofUL7. One exemplary mutation is a deletion of a adenine (e.g., theadenine at nucleotide position 98125 of the ILTC genome) from the codon,but a person of ordinary skill in the art can readily conceive of othermutations that could cause a frame shift at Phe104—or any other site—ofUL7.

A frame shift mutation in UL9 may include, for example, a frame shiftintroduced at PGlu771 such as may be produced by a deletion of anucleotide or an addition of a nucleotide in the codon for Glu771 ofUL9. One exemplary mutation is a deletion of a adenine (e.g., theadenine at nucleotide position 94565 of the ILTV genome) from the codon,but a person of ordinary skill in the art can readily conceive of othermutations that could cause a frame shift at Glu771—or any other site—ofUL9.

In some embodiments, an attenuated ILTV can include a geneticmodification to the UL50 coding region without necessarily possessingany of the other genetic modifications shown in Table 11. Moreover, insome embodiments, the genetic modification to UL50 may differ from thethymine deletion identified in Table 11, while still resulting inattenuation of the resulting genetically-modified ILTV.

The attenuated ILTV, when administered to a subject, can decrease thelikelihood, extent, and/or severity of infection of the subject by avirulent ILTV compared to a comparable unvaccinated control subject. Insome cases, administering the attenuated ILTV can result in increasedweight gain and/or a decrease in the severity of one or more signs ofinfection by a virulent ILTV. Exemplary signs of infection by a virulentILTV include, for example, conjunctival swelling and/or reddening,presence of an ocular and/or nasal (nares) exudate, nasal sinusswelling, labored breathing, coughing, and in severe cases, a bloodyexpectoration.

Thus, in another aspect, this disclosure describes a method thatinvolves administering the attenuated infectious laryngotracheitis virusdescribed herein to a subject having or at risk of having an ILTVinfection.

Thus, treating a condition can be prophylactic or, alternatively, can beinitiated after the subject exhibits one or more symptoms or clinicalsigns of the condition. As used herein, the term “sign” or “clinicalsign” refers to an objective physical finding relating to a particularcondition capable of being found by one other than the subject; the term“symptom” refers to any subjective evidence of disease or of a subject'scondition. Treatment that is prophylactic—e.g., initiated before asubject manifests a symptom or clinical sign of the condition such as,for example, while an infection remains subclinical—is referred toherein as treatment of a subject that is “at risk” of having thecondition. As used herein, the term “at risk” refers to a subject thatmay or may not actually possess the described risk. Thus, for example, asubject “at risk” of having an infectious condition is a subject presentin an area where other individuals have been identified as having theinfectious condition and/or is likely to be exposed to the infectiousagent. A subject may be “at risk” even if the subject has not yetmanifested any detectable indication of infection by a virulent ILTV andregardless of whether the animal may harbor a subclinical amount of avirulent ILTV.

Accordingly, the attenuated ILTV can be administered to a subjectbefore, during, or after the subject first comes in contact with avirulent ILTV. Treatment initiated after the subject first exhibitscomes in contact with a virulent ILTV may result in decreasing theseverity of symptoms and/or clinical signs of the condition, completelyresolving the condition, and/or decreasing the likelihood ofexperiencing clinical evidence of the condition compared to a subject towhich the attenuated ILTV has not been administered.

The method includes administering an effective amount of the attenuatedILTV to a subject exposed to, or at risk of being exposed to ILTV. Inthis aspect of the invention, an “effective amount” is an amounteffective to reduce, limit progression, ameliorate, or resolve, to anyextent, the symptoms or clinical signs related to infectiouslaryngotracheitis.

The attenuated ILTV described herein may be formulated in a compositionalong with a “carrier.” As used herein, “carrier” includes any solvent,stabilizer, dispersion medium, vehicle, coating, diluent, antibacterial,and/or antifungal agent, isotonic agent, absorption delaying agent,buffer, carrier solution, suspension, colloid, and the like. The use ofsuch media and/or agents for pharmaceutical active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the attenuated ILTV described herein, its use in atherapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the attenuated ILTV withoutcausing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained.

The attenuated ILTV may be administered to any suitable subject such as,for example, chickens, turkeys, ducks, pheasants, and peafowl.

In the preceding description, particular embodiments may be described inisolation for clarity. Unless otherwise expressly specified that thefeatures of a particular embodiment are incompatible with the featuresof another embodiment, certain embodiments can include a combination ofcompatible features described herein in connection with one or moreembodiments.

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1 Generating P32 37C

The P32 37C virus was serially passaged 32 times in primary chickenembryo kidney cells (CEKC), incubated at 37° C.

Kidney cells were prepared from 17-20 day old embryos.

The amounts indicated here are for preparing kidney cells from 10-15embryos. Eggs were sprayed with a disinfectant (BIOGUARD, BioguardHygiene Solutions Ltd., Northampton, UK) and allowed to dry. Kidneyswere dissected from the embryos, washed in Phosphate Buffered Saline(PBS) without calcium or magnesium (1.60 g Na₂HPO₄, 0.51 g KH₂PO₄, 7.30g NaCl in 1 L DDH₂O). The kidneys were dissociated and treated withtrypsin solution (0.25% trypsin in modified PBS+0.02% EDTA, pH 7.4-7.6)and stirred slowly for 15-20 minutes at 37° C. The supernatant wascollected by pouring through a gauze-covered funnel into a graduatedcentrifuge tube with 5 ml of cold (ice bath-cooled) heat-inactivatedcalf serum. The remaining pellet was retrypsinized with fresh trypsinsolution and the process repeated two more times. The collectedsupernatant was centrifuges at 1500 RPM for 10 minutes. The pelletedkidney cells were resuspended in 3-6 ml minimal essential medium (MEM),then added to MEM with 10% heat-inactivated fetal calf serum.

Cells were counted in a hemacytometer by resuspending in a known amountof media. 2 ml of cells (2.5×10⁶ cells/ml) were plated on a 35 mm²plate, which formed a monolayer in 1-2 days. When the monolayer formed,the cells were ready for inoculation.

1 mL of starting virus was used to infect one 25 mL flask with 80%confluent monolayer of CEKC's. The inoculated CEKC were incubated at 37°C. ILTV cytopathic effect (CPE) known as syncytia were observed in theCEKC at two days after inoculation. The cells and culture media wereharvested and repassaged serially for 32 passages at an incubationtemperature of 37° C. A seed stock of the virus was then prepared forfurther experimentation.

Generating P32 30C

The P32 30C virus was serially passaged 32 times in primary chickenembryo kidney cells (CEKC). Cells were prepared as described above forthe preparation of the P32 37C virus, except that the inoculated CEKCwere incubated at 30° C. ILTV cytopathic effect (CPE) known as syncytiawere observed in the CEKC at 3-4 days after inoculation. The cells andculture media were harvested and repassaged serially for 32 passages atan incubation temperature of 30° C. A seed stock of the virus was thenprepared for further experimentation.

Assessment of Vaccines

One-day-old, female commercial broiler chickens (Moyer's Chicks, Inc.,Quakertown, Pa.) were assigned to one of five treatment groups shown inTable 9.

TABLE 9 Group Immunizing Vacc. No. of Birds No. Virus Titer (Oral/ED) 1TCO Vaccine^(A) 10^(3.0) 10/5 2 CEO Vaccine^(B) 10^(4.0) 10/5 3 P32 30C.^(C) 10^(3.0) 10/5 4 P32 37 C.^(D) 10^(3.0) 10/5 5 Negative ControlSham 10/5 ^(A)TCO = Tissue culture origin (LT-IVAX, Merck Animal Health,Summit, NJ). ^(B)CEO = Chicken embryo origin (TRACHIVAX, Merck AnimalHealth, Summit, NJ). ^(C)P32 30 C. = 32 tissue culture passagesperformed at 30° C. ^(D)P32 37 C. = 32 tissue culture passages performedat 37° C.

The night prior to vaccination at 14 days of age, water was withheldfrom the birds. On the afternoon prior to vaccination, all birds wereweighed. Birds in the oral vaccinated groups received 1.5 mL of watercontaining one drop of vaccine (0.03 ml) by hand using a blunt needleand syringe. Vaccination occurred slowly to ensure proper uptake by thebird. Each bird was held vertically, beak pointed slightly toward theceiling to mimic drinking from a nipple. Each bird in the correspondingeye drop vaccine group was vaccinated by placing one drop (0.03 ml) ofinoculum directly into the conjunctival sac.

Birds were observed daily for signs of vaccine reaction (conjunctivitis,nasal discharge). Six days post vaccination, all birds were weighed.Oropharyngeal swabs were collected from all birds to determine the levelof ILTV present in the oral cavity by real-time PCR (Callison et al.,2007. J. Virol. Methods 139:31-38). Two birds from each water vaccinatedgroup and the negative control group were euthanized. Trachea and eyelidwere collected for evaluation of microscopic pathology.

Fourteen days post vaccination, all birds were weighed, serum sampleswere collected from all birds prior to challenge with the USDA challengestrain of ILTV (10^(3.5)/0.1 ml). ILTV serum antibody titers wereevaluated using a commercial ELISA kit (Synbiotics Corp., Kansas City,Mo.). Five days post challenge birds were weighed and evaluated forsigns of disease. Trachea and eyelid were collected after euthanizingvia cervical dislocation.

Results are shown in Tables 1-3.

Example 2 Generating Clone 5-7 from P 32 30C Using the Limit DilutionProcedure

Serial 10-fold dilutions (10⁻¹-10⁻⁶) of the P32 30C virus were preparedand inoculated onto CEKC. The inoculated cells were incubated at 30° C.The CEKC were observed microscopically daily for syncytia. Cells andmedia were harvested from the highest dilution showing syncytia. Aculture plate at the 10⁻⁵ dilution, 7^(th) replicate plate met thecriteria and the cells and media were harvested. A seed stock wasprepared for further experimentation.

Assessment of Vaccines

Female broiler type chickens (Moyer's Chicks, Inc., Quakertown, Pa.).Chicks were placed in isolation in groups of 13 birds. At 20 days ofage, birds were water deprived for at least two hours prior toinoculation. The following day, at 21 days of age, birds were randomlyregrouped, assigned to treatment groups (Table 10) and necktagged foridentification. All birds were weighed. Birds with bodyweights greaterthan or less than 2SD of the group mean weight were used in the swabcontrol groups. The negative control birds were placed in isolationprior to handling any viruses. Birds in the negative control/oral groupreceived 1 mL of water. No other sham inoculations were performed.

TABLE 10 Cage No. of Female No. Treatment Treatment Broilers 1 NegativeChallenge Controls 5 Control Oral 5 Ocular 5 Swab Controls 2 2 10⁻¹ Oral5 Ocular 5 3 10⁻² Oral 5 Ocular 5 Swab Controls 2 4 10⁻³ Oral 5 Ocular 5Swab Controls 2 5 10⁻⁴ Oral 5 Ocular 5 Swab Controls 2 6 10⁻⁵ Oral 5Ocular 5 Swab Controls 2 7 10⁻⁶ Oral 5 Ocular 5 Swab Controls 2

Inoculation:

Prior to inoculation with the Clone 5-7 dilutions, all birds wereweighed and blood samples were collected from 10 birds. Six 8-ml tubeswere filled with 4.5 ml of sterile water (pH 7.0—spring water). Tubeswere used for to create the inoculum dilutions (10⁻¹ to 10⁻⁶). Six10-fold dilutions of the Clone 5-7 ILTV were created by placing 0.5 mlof virus in 4.5 ml of sterile water (pH 7.0—spring water). 0.5 ml of the10⁻¹ dilution was then be placed into 4.5 ml of sterile water (pH7.0—spring water) to create the 10⁻² and repeated until the 10⁻⁶dilution was created. 0.5 ml were removed from the 10⁻⁶ dilution anddiscarded. All dilution tubes contained 4.5 ml of the virus/waterdilution.

Two separate tubes were marked as either for ocular or oral inoculation.The ocular tube contained at least 1 ml of the given dilution. The oraltube contained 0.7 ml of inoculum and 7 ml of water. These tubes wereheld on ice until used. Ocular treatment birds received 0.1 ml (100 μl)by eyedrop. The oral treatment birds received 1 ml of the water/viruspreparation. Swab controls received 0.1 ml (100 μl) of a given vaccinetreatment via eyedrop. These birds were used to assess viral load in thetrachea by real-time PCR.

6 DPI Swab and Weigh:

Birds were swabbed at six day post-inoculation (dpi) to determine viralload by real time PCR in the oral cavity of the ocular and oralchallenged groups. Tracheal and oral swabs were collected from the swabcontrols to evaluate viral load at both swabbing locations by real-timePCR (Callison et al., 2007. J. Virol. Methods 139:31-38). Body weightswere also collected to determine the effect of infection on weight gain.Swabs were collected and placed in 2 ml of sterile Tryptose PhosphateBroth (Difco, BD Biosciences, San Jose, Calif.).

14 DPI-Challenge:

All birds were weighed and bled prior to challenge. The five “challengecontrols” in cage 1 were used to verify infection of the broilers usingchallenge dose of 10³ per bird. After challenge, the challenge controlswere relocated among the 10⁻⁵ and 10⁻⁶ treatment groups (cages 6 and 7).All birds in cages 2-7 received a challenge of 10³ EID₅₀ of the USDAILTV challenge strain per 0.1 ml divided between the intraocular (onedrop) and oral (remaining volume up to 0.1 ml) routes of inoculationusing a 200 μl pipette.

Birds were evaluated daily for signs of clinical disease for a period of10 days post challenge (DPC). At 6 DPC, birds were weighed to evaluateprotection based upon suppression of daily weight gain compared tonon-challenged controls. At 10 DPC, birds were weighed and evaluated forsigns of clinical disease. All surviving birds were bled to evaluateILTV antibody status following challenge. ELISA titers were evaluated todetermine if they might be indicative of lack of protection or a boosterresponse. The experiment was terminated after 10 DPC.

Results are shown in Tables 4-6.

Genetic Signature of Clone 5-7

Approximately 500 μL of the Clone 5-7 TCO supernatant was treated withDNase and the viral DNA was extracted using the DNeasy 96 Blood & TissueKit (Qiagen, Inc., Valencia, Calif.) according the recommendedprocedures. A DNA library was generated using the NuGen OVATION ultralowlibrary system (NuGEN Technologies, Inc., San Carlos, Calif.). Thelibraries were sequenced on an Illumina Genome Analyzer IIx (Illumina,Inc., San Diego, Calif.) in a paired-end, 101 cycle run. Library readswere assembled to a reference ILTV genome using CLCWorkbench (CLC bio,Inc., Germantown, Md.). Variant detection within the Clone 5-7 assembledgenome and the impact of the detected variation on amino acid sequencewas performed within CLCWorkbench (CLC bio, Inc.,).

TABLE 11 ILTV Clone 5-7 Genetic Signature vs. Commercial TCO VaccineDepth of Coding Nt TCO ILTV Clone Non-synonymous Amino Acid Coverage atFrequency of Region Position# Vaccine Nt 5-7 Nt or Synonymous ChangePosition Mutation (%) NC 1160 A — Not Applicable Not Applicable 20203.81 NC 104594 A — Not Applicable Not Applicable 4493 93.08 NC109606{circumflex over ( )}109607 — G Not Applicable Not Applicable 198172.19 NC 109614 T — Not Applicable Not Applicable 980 6.22 NC109616{circumflex over ( )}109617 — G Not Applicable Not Applicable 16893.02 NC 109622 G — Not Applicable Not Applicable 1450 6.55 ORF B 23030 T— Non-synonymous Phe54fs* 5030 6.92 ORF F 5377{circumflex over ( )}5378— G Non-synonymous Glu233fs 5489 3.06 UL36 53320 A G Non-synonymousVal2300Ala 4082 16.39 UL37 62826 T C Synonymous Not Applicable 4129 3.44UL50 15577 T — Non-synonymous Asn10fs 4582 92.45 UL7 98125 A —Non-synonymous Phe104fs 4737 5.3 UL9 94565 A — Non-synonymous Glu771fs4339 5.49 *fs = frame shift #SEQ ID NO: 1, Genbank accession no.JN580312

Example 3

Female specific pathogen free (SPF) leghorn chickens were obtained atfive weeks of age from Charles-River SPAFAS (Norwich, Conn.). Birds wereperiodically obtained in lots of 15 or 30.

Passage 1

For each passage, birds were assigned to one of two groups: DirectInoculated (10 birds) or Challenge Controls (5 birds) (Table 7 and Table8). Each bird was tagged according to treatment group. Clone 5-7 wasdiluted 1:100 and administered via eye-drop (0.1 mL/bird) to each of the10 birds in Cage 1. On day 5 post challenge (PC), an oropharyngealswabbing, culminating in the swabbing of the upper tracheal opening,were collected from each bird and placed into two swab tubes (5 swabs ineach tube), each containing 2.5 mL of Tryptose Phosphate Broth (Difco,BD Biosciences, San Jose, Calif.) with antibiotics (penicillin (100I.U.) and streptomycin (100 μg/ml), with fungizone (2.5 μg/ml)). Tubeswere held at −80° C. until need for subsequent challenge.

Birds were observed and scored daily for disease signs (conjunctivitis,labored breathing/gasping, mortality) through day 10 post-directinoculation. Birds were scored as 0=normal, 1=diseased (respiratorysigns, swollen eye), or 2=dead. Swab tubes were screened for thepresence of ILTV genome by real-time PCR (Callison et al., 2007. J.Virol. Methods 139:31-38).

Fourteen days post inoculation, the Direct Inoculates (10 birds) and theChallenge Controls (5 birds) were bled to determine ILTV serum antibodystatus by enzyme-linked immunosorbent assay (ELISA) (Synbiotics Corp.,Kansas City, Mo.). All birds were moved to the Clone 5-7 Challenge Cageand challenged via eye-drop with the USDA ILTV Challenge Virus(10^(3.5)/bird). All birds were evaluated through 10 days PC for diseasesigns using the same scoring scale defined above.

Passage 2 to Passage 5

On the defined day of challenge, the two swab tubes collected five dayspost challenge with the previous passage were thawed at room temperatureand centrifuged to pellet any debris. An aliquot of 0.5 ml from eachtube was taken and pooled to create the inoculum for birds. The 10 birdsin the direct inoculation group received 0.1 ml of the undiluted swabpool material via eye-drop. On day 5 post challenge, an oropharyngealswabbing (culminating in the swabbing of the upper tracheal opening) wascollected from each bird and placed into two swab tubes (5 swabs in eachtube) each containing 2.5 ml of Tryptose Phosphate Broth (Difco, BDBiosciences, San Jose, Calif.) with antibiotics (penicillin (100 I.U.)and streptomycin (100 μg/ml) with fungizone (2.5 μg/ml)). Tubes wereheld at −80° C. until need for subsequent challenge.

Birds were observed and scored daily for disease signs (conjunctivitis,labored breathing/gasping, mortality) through day 10 post-directinoculation. Birds were scored as described for Passage 1. Swab tubeswere screened for the presence of ILTV genome by real-time PCR asdescribed for Passage 1.

Fourteen days post inoculation, the Direct Inoculates (10 birds) and theChallenge Controls (5 birds) were bled to determine ILTV serum antibodystatus as described for Passage 1. All birds were challenged viaeye-drop and evaluated through 10 days post challenge as described forPassage 1.

The inoculation procedure described above was repeated until ILTV was nolonger detected by real-time PCR, the birds were susceptible tochallenge, or a total of 5 bird passages had been completed.

Results are shown in Tables 7 and 8.

As used herein, the term “and/or” means one or all of the listedelements or a combination of any two or more of the listed elements; theterms “comprises” and variations thereof do not have a limiting meaningwhere these terms appear in the description and claims; unless otherwisespecified, “a,” “an,” “the,” and “at least one” are used interchangeablyand mean one or more than one; and the recitations of numerical rangesby endpoints include all numbers subsumed within that range (e.g., 1 to5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference in their entirety. In theevent that any inconsistency exists between the disclosure of thepresent application and the disclosure(s) of any document incorporatedherein by reference, the disclosure of the present application shallgovern. The foregoing detailed description and examples have been givenfor clarity of understanding only. No unnecessary limitations are to beunderstood therefrom. The invention is not limited to the exact detailsshown and described, for variations obvious to one skilled in the artwill be included within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

1. An attenuated infectious laryngotracheitis virus (ILTV) comprising:at least one genetic modification listed in Table
 11. 2. The attenuatedinfectious laryngotracheitis virus of claim 1 wherein the mutationcomprises a deletion of a thymine at nucleotide position 23030 of SEQ IDNO:1.
 3. The attenuated infectious laryngotracheitis virus of claim 2wherein the mutation introduces a frame shift at Phe 54 of ORF B.
 4. Theattenuated infectious laryngotracheitis virus of claim 1 wherein themutation comprises an insertion of a guanine between nucleotide position5377 and 5388 of SEQ ID NO:1.
 5. The attenuated infectiouslaryngotracheitis virus of claim 4 wherein the mutation produces a frameshift at Glu233 of ORF F.
 6. The attenuated infectious laryngotracheitisvirus of claim 1 wherein the mutation comprises a substitution of aguanine for an adenine at nucleotide position 53320 of SEQ ID NO:1. 7.The attenuated infectious laryngotracheitis virus of claim 6 wherein themutation produces a substitution of an alanine for a valine at position2300 of UL36.
 8. The attenuated infectious laryngotracheitis virus ofclaim 1 wherein the mutation comprises a deletion of a thymine atnucleotide position 15577 of SEQ ID NO:1.
 9. The attenuated infectiouslaryngotracheitis virus of claim 8 wherein the mutation produces a frameshift at Asn10 of UL50.
 10. The attenuated infectious laryngotracheitisvirus of claim 1 wherein the mutation comprises a deletion of an adenineat nucleotide position 98125 of SEQ ID NO:1.
 11. The attenuatedinfectious laryngotracheitis virus of claim 10 wherein the mutationproduces a frame shift at Phe104 of UL7.
 12. The attenuated infectiouslaryngotracheitis virus of claim 1 wherein the mutation comprises adeletion of a adenine at nucleotide position 94565 of SEQ ID NO:1. 13.The attenuated infectious laryngotracheitis virus of claim 12 whereinthe mutation produces a frame shift at Glu771 of UL9.
 14. The attenuatedinfectious laryngotracheitis virus of claim 1 wherein oraladministration of the virus to a subject produces increased weight gainand/or decreased sign of infectious laryngotracheitis disease followingchallenge with a virulent ILTV, compared with an unvaccinated controlsubject.
 15. A method comprising: administering the attenuatedinfectious laryngotracheitis virus of claim 1 to a subject having or atrisk of having an ILTV infection.
 16. (canceled)
 17. A method ofproducing a live attenuated infectious laryngotracheitis virus (ILTV),the method comprising serially passaging the ILTV in culture at atemperature no greater than 35° C.
 18. The method of claim 17 whereinILTV is passaged for at least five passages.
 19. The method of claim 17wherein the ILTV is passaged in culture at a temperature of from 28° C.to 31° C.